Avaliação da sazonalidade reprodutiva sobre os mecanismos da estocagem de espermatozoides e os parâmetros espermáticas da cascavel neotropical Crotalus durissus (Squamata: Viperidae)

Abstract

The Neotropical rattlesnake (Crotalus durissus) is a medical-importance snake species from the Viperidae family, with a wide distribution throughout Latin America. Males of C. durissus present a seasonal spermatogenic cycle, which begins in spring and reaches its peak in the summer. During autumn and winter males pass through testicular regression. Mating was recorded in the autumn, therefore, due to dissociation between spermatogenesis and copula, sperm storage is obligatory. However, the seminal parameters, the gamete viability, and the time of sperm storage are not known. In the present study, we aimed to investigate morphofunctional aspects of spermatozoa and the mechanisms of gamete storage in Crotalus durissus throughout its reproductive cycle. In total, 46 animals were collected in the state of Minas Gerais, southeastern Brazil. The animals were euthanized, and the reproductive system organs were collected. We carry out seminal analyses, sperm morphometry, standardization of sperm morphology, sperm lifespan, cell marking to estimate gamete storage time, immunohistochemistry, immunofluorescence, and DNA integrity analysis. The spermatozoa of C. durissus are divided into head, midpiece and tail. The head is elongated and slightly curved. The midpiece is the largest segment of the spermatozoa, occupying about 56% of its total length. Finally, there is a tail, which is responsible for the movement of the gamete. There is a variation in the length of the acrosome and sperm head, which are larger in animals with regressed testes. This characteristic might indicate that the storage time in the ductus deferens propitiates the maturation of gametes. There is no variation in sperm motility between animals with different testicular conditions. Therefore, there must be mechanisms that protect the gametes until copulation. However, there are more live sperm during the period of spermatogenic activity when compared to animals undergoing testicular regression. Despite this, sperm vitality during testicular regression is still over 70%. We observed that rattlesnake sperm kept at low temperatures are viable for at least seven days, maintaining high levels of vitality. This result demonstrates the high resistance of sperm and opens the door to assisted reproductive technologies. The epithelium of the ductus deferens reacted positively to the expression of the androgen receptor and aquaporin 9. These findings demonstrate that the synthesis of secretions in the ductus deferens is possibly stimulated by androgens and that, through the expression of aquaporins, there is fluid reabsorption. This may be one of the mechanisms that guarantee gamete viability. We were able to detect bromodeoxyuridine-labeled sperm only in animals euthanized after 40 days of injection, corroborating the duration of the spermatogenic cycle. Using the cell death marker Caspase-3, we observed more sperm undergoing apoptosis during the period of testicular regression. In the same way, we observed more spermatozoa with fragmented DNA in animals with regressed testes.